This invention was made in part with Government funding, and the Government therefore has certain rights in the invention.
The invention relates to the diagnosis and treatment of conditions associated with cell proliferation.
The control of cell proliferation is a fundamental issue in medicine. Cell proliferation is regulated by the formation and dissociation of multiple protein complexes, the components of which often use post-translational modifications as an additional control mechanism. Many diseases result from inappropriate cell proliferation, including cancer. Methods and reagents to modulate cell proliferation would have the potential to yield new treatments for cancer, major opportunistic infections, immune disorders, certain cardiovascular diseases, and inflammatory disorders.
In general, the invention features a substantially pure nucleic acid (for example, genomic DNA, cDNA, synthetic DNA, mRNA, or antisense RNA) encoding a p54S6K or p85S6K polypeptide, as defined below.
In a preferred embodiment, the substantially pure nucleic acid encoding a p54S6K or p85S6K polypeptide is mammalian DNA. More preferably, the substantially pure nucleic acid encoding a p54S6K or p85S6K polypeptide is human DNA. In another embodiment, the invention features a DNA sequence substantially identical to, or which hybridizes with high stringency to, the DNA sequence shown in FIG. 1A or 5 (SEQ ID NO: 1).
In another embodiment, the invention features DNA encoding fragments of p54S6K or p85S6K polypeptides. In preferred embodiments, the fragments include the C-terminal and N-terminal regions of p54S6K or p85S6K that are distinct from p70S6k, the catalytic domain of p54S6K or p85S6K, the linker domain, the proline rich domain, or the acidic region of p54S6K or p85S6K. In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54S6K (SEQ ID NOs: 4-10). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85S6K (SEQ ID NOs: 10-15). In another embodiment, the invention features nucleic acids that bind with high stringency to SEQ ID NOs: 1 or 2.
In another aspect, the invention features a substantially pure polypeptide having a sequence substantially identical to SEQ ID NOs: 2 or 3. In a preferred embodiment, the substantially pure polypeptide has an acidic region at its N-terminus and a central catalytic region. In another the preferred embodiment, the substantially pure polypeptide has a post-translational modification
In other aspects of the invention, a substantially pure p54S6K or p85S6K polypeptide has one or more amino acids changed by natural or artificial means, the p54S6K or p85S6K polypeptide is p54S6K KR or p85S6K KR, or the p54S6K or p85S6K polypeptide is a fusion with a heterologous polypeptide, for example, glutathione-S-transferase (GST) or influenza hemagglutinin (HA), relative to the wild-type p54S6K or p85S6K sequence shown in FIG. 1A or FIG. 5.
In another aspect, the invention features fragments of p54S6K or p85S6K polypeptides. In a preferred embodiment, the fragment is a peptide comprising a C-terminal sequence of p54S6K or p85S6K that is distinct from p70S6k or p85S6K. In another embodiment, the fragment comprises a N-terminal sequence of p54S6K or p85S6K that is distinct from p70S6k. In another embodiment, the fragment comprises the catalytic domain of p54S6K or p85S6K. In another embodiment, the fragment comprises the acidic region of p54S6K or p85S6K In another embodiment, the fragment comprises the linker domain of p54S6K or p85S6K. In another embodiment, the fragment comprises the proline rich region of p54S6K or p85S6K. In another embodiment, the fragment contains a post-translational modification of p54S6K or p85S6K. In yet another embodiment, the fragment is a fragment of p54S6K KR or p85S6K KR. In still another embodiment, the fragment is fused to a heterologous polypeptide. In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54S6K (SEQ ID NOs: 4-9). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85S6K (SEQ ID NOs: 10-15).
In another aspect, the invention features an antibody that specifically binds to a p54S6K or p85S6K polypeptide, a p54S6K or p85S6K polypeptide fragment, or a post-translational modification of a p54S6K or p85S6K polypeptide. In a preferred embodiment, the antibody is Ab#167.
In another aspect, the invention features a cell line having a genetically engineered null mutation in a p54S6K or p85S6K gene. In a preferred embodiment, the cell line is an embryonic stem cell line. If the cell is in a mammal, the mammal is preferably a non-human.
In another aspect, the invention features a cell line, genetically engineered to overexpress a p54S6K or p85S6K polypeptide. In a preferred embodiment, the cell line is a tumor cell line.
In another aspect, the invention features a non-human transgenic animal, or embryo thereof, with a knockout mutation in the p54S6K or p85S6K gene. In a related aspect, the invention features a non-human transgenic animal with additional copies of p54S6K or p85S6K nucleic acids added to its genome. In a preferred embodiment of these aspects, the non-human transgenic animal is a rodent, more preferably a mouse. In a preferred embodiment the animal has a knockout mutation in both genes.
In other aspects, the invention features methods of identifying a compound which modulates, or whose activity is modulated by, p54S6K or p85S6K biological activity involving: a) providing a cell expressing a p54S6K or p85S6K polypeptide, or a lysate from the cell expressing a p54S6K or p85S6K polypeptide, or a transgenic animal expressing a p54S6K or p85S6K polypeptide; b) exposing the cell, lysate, or transgenic animal to the test compound; c) assaying for a modulation in p54S6K or p85S6K biological activity; and d) comparing the modulation in p54S6K or p85S6K biological activity to that of a cell, lysate, or transgenic animal which did not receive the test compound, wherein a modulation of p54S6K or p85S6K biological activity identifies a test compound. In a preferred embodiment the biological activity is assayed by measuring p54S6K or p85S6K phosphorylation, p54S6K or p85S6K kinase activity, p54S6K or p85S6K polypeptide or nucleic acid levels, or cell proliferation. In another preferred embodiment, p54 or p85S6K biological activity is measured by using S6 or BRCA1 as a substrate. In other embodiments, the cell is induced or genetically engineered to express the p54S6K or p85S6K polypeptide. In a preferred embodiment the cell is a tumor cell and/or the p54S6K or p85S6K polypeptide is p54S6K KR or p85S6K KR. In another preferred embodiment, the test compound is BRCA1. In a related aspect, the cell may express an altered p54S6K or p85S6K, as described herein before (e.g., a fusion, fragment, etc.),
In other embodiments, the cell is exposed to a stimulus which could include serum or insulin. In other embodiments the cell is exposed to an inhibitor which could include rapamycin or wortmannin.
In another aspect, the invention features a method of diagnosing an increased likelihood of a cell proliferative disease in a subject. The method includes detecting the level of p54 or p85S6K gene expression in the subject. In preferred embodiments of this aspect, detection of the level of p54S6K or p85S6K gene expression is done by nucleic acid hybridization, including Northern blotting, Southern blotting, Western blotting, far-Western blotting, or DNA-mRNA hybridization. In another embodiment the subject is a human.
In a final aspect, the invention features a therapeutic composition comprising as an active ingredient a p54S6K or p85S6K polypeptide or nucleic acid, the active ingredient being formulated in a physiologically acceptable carrier. In a preferred embodiment, the p54S6K or p85S6K nucleic acid is in the context of a vector, more preferably a gene therapy vector.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the substances that naturally accompany it. In the case of DNA, it would mean DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. In the case of RNA, it would include mRNA free of associated proteins and nucleic acids.
By xe2x80x9cp54S6Kxe2x80x9d is meant any gene or polypeptide, which is not p70S6k, which hybridizes with high stringency to SEQ ID NO: 1 or and has p54S6K biological activity.
By xe2x80x9cp85S6Kxe2x80x9d is meant any gene or polypeptide, which is not p70S6k, which hybridizes with high stringency to a gene encoding the polypeptide of SEQ ID NO: 3 and has p85S6K biological activity.
By xe2x80x9chigh stringencyxe2x80x9d is meant conditions that are commonly understood in the art as stringent. An exemplary set of high stringency conditions include a temperature of 60-70xc2x0 C., (preferably about 65xc2x0 C.) and a salt concentration of 0.70 M to 0.80 M (preferably about 0.75 M). Further exemplary conditions include, hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50xc2x0 C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C.; or (3) employ 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M sodium citrate), 5xc3x97Denhardt""s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2xc3x97SSC and 0.1% SDS. Further examples of stringent conditions can be found in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), Cold Spring Harbor Press, 1989, or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1994).
By xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide, or constituting a non-naturally occurring polypeptide.
By xe2x80x9csubstantially pure p54S6K polypeptidexe2x80x9d is meant a p54S6K polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, p54S6K polypeptide. A substantially pure p54S6K polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an p54S6K polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cells from which is naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure p54S6K polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure p85S6K polypeptidexe2x80x9d is meant a p85S6K polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, p85S6K polypeptide. A substantially pure p85S6K polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an p85S6K polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cells from which is naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure p85S6K polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid sequence (for example, the amino acid sequence described herein) or nucleic acid sequence (for example, the nucleic acid sequence described herein). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 75 nucleotides, and more preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody which recognizes and binds a protein, but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes protein.
By xe2x80x9cpost-translational modificationxe2x80x9d is meant any changes to the polypeptide during or after synthesis. These modifications may include phosphorylation and glycosylation. Post-translational modifications may be naturally occurring (such as during synthesis within a cell), or artificially generated (such as by recombinant or chemical means).
By xe2x80x9cnull cell linexe2x80x9d is meant a cell line that does not express a p54S6K or does not express a p85S6K polypeptide or which expresses a p54S6K or p85S6K polypeptide fragment having decreased biological activity. Preferably the cell line lacks detectable biological activity for said polypeptide. The cell line may be created by genetic engineering such that there is a mutation that makes a p54S6K or p85S6K polypeptide that has no detectable biological activity, or prevents the cell from making a p54S6K or p85S6K polypeptide.
By xe2x80x9coverexpressingxe2x80x9d is meant a cell line that expresses a p54S6K or p85S6K polypeptide or a p54S6K or p85S6K polypeptide fragment at a level at least 10% higher than the level of endogenous expression. This would include cells that express p54S6K or p85S6K either transiently, or stably incorporated into their genome. It would also include both prokaryotic and eukaryotic cells.
By xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate p54S6K or p85S6K biological activity, by employing one of the assay methods described herein. Compounds may include, for example, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9cmodulatesxe2x80x9d is meant changes, either by increase or decrease.
By xe2x80x9cp54S6K biological activityxe2x80x9d is meant any activity of p54S6K found in cells. This includes the levels of p54S6K nucleic acids or polypeptides, p54S6K phosphorylation, p54S6K kinase activity towards a variety of substrates, the inhibition of p54S6K activity by natural or artificial compounds, and the effect of p54S6K on substrates. This also includes the effect of p54S6K on cell proliferation and cell cycle.
By xe2x80x9cp85S6K biological activityxe2x80x9d is meant any activity of p85S6K found in cells. This includes the levels f p85S6K nucleic acids or polypeptides, p85S6K phosphorylation, p85S6K kinase activity towards a variety of substrates, the inhibition of p85S6K activity by natural or artificial compounds, and the effect of p85S6K on substrates. This also includes the effect of p85S6K on cell proliferation and cell cycle.
By xe2x80x9cexposingxe2x80x9d is meant allowing contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a compound.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome.
By xe2x80x9cknockout mutationxe2x80x9d is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation. Preferably, the mutation is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
By xe2x80x9cassayingxe2x80x9d is meant analyzing the effect of a treatment or exposure, be it chemical or physical, administered to whole animals or cells. The material being analyzed may be an animal, a cell, a lysate or extract derived from cell, or a molecule derived from a cell. The analysis may be, for example, cell proliferation, cell morphology, or tumor formation. Techniques used in the analysis may include SDS polyacrylamide gel electrophoresis, in vitro kinase reactions, immunoprecipitation, etc.
By xe2x80x9csubstratexe2x80x9d is meant a molecule whose activity is modulated by p54S6K or p85S6K in vivo or in vitro. Substrates may include S6 and BRCA1. Preferably, a substrate is a molecule that has a phosphorylation state which is modified by p54S6K or p85S6K protein.
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a p54S6K or p85S6K gene.
By xe2x80x9cdetecting the level of p54S6K gene expressionxe2x80x9d is meant detecting the levels of p54S6K polypeptides or nucleic acids. The detection would be relative to normal samples from patients without the cell proliferative disease.
By xe2x80x9cdetecting the level of p85S6K gene expressionxe2x80x9d is meant detecting the levels of p85S6K polypeptides or nucleic acids. The detection would be relative to normal samples from patients without the cell proliferative disease.
The invention provides methods and reagents for the diagnosis and treatment of diseases caused by inappropriate cell proliferation. These disorders can be treated, using the methods described herein, in a variety of ways including small molecules, gene therapy, antisense oligonucleotides and protein replacement. Additionally, they can have potential diagnostic/disease management applications as prognostic markers or predisposition indicators of certain cancers. Other features and advantages of the invention will be apparent from the detailed description of the invention, and from the claims.